Agricultural waste products have developed into massive accumulations throughout the world and are, at best, civic nuisances unless they are converted, somehow, to beneficial utility. Vegetative wastes such as corn cobs are ground into such items as litter absorbents, see for example U.S. Pat. No. 6,092,302, and, even after such manner of use, contribute additionally, again, to the waste stream. At the same time, utilization of biochemical machinery in the field of nanotechnology to achieve practical purposes have tended to focus on fabrication of novel architectures that range from Bucky Balls to metal clusters on DNA scaffolds (see, for example, U.S. Pat. No. 6,730,537; and Shen, G.-R., Cheng, Y.-T., and Tsai, L.-N. 2005. Synthesis and Characterization of Ni—P-CNT's Nanocomposite Film for MEMS Applications. IEEE Transactions on Nanotechnology 4(5):539-547), while purposeful in vivo applications are limited.
The specific focus of the present invention is the ubiquitous cell wall component, pectin. The present invention generally relates to three fundamental designs of pectins: (1) As surface coatings; (2) As novel compositions of matter; and 3) As components that are integrated and transported systemically to the benefit of an entire organism.
In live systems, pectin is involved in the development of all green plants as a key molecule of cell walls. Under the mild conditions of the present invention, cations and saccharides are selected for their similarities to those found in pectin, insuring that they will bind to pectins of the living cell. Natural pectin can be changed, thereby, in size, charge, and function. A new molecule is created as it bonds with cations and saccharides.
Pectins are complexes of heterosaccharides, of which their polygalacutronic acid (PGA) junction zones are characterized by ionic bonds to di-cationic calcium (Ca2+). Thus, pectins are Ca2+-bearing macromolecules physically measuring in a range of approximately 13 to 45 nm and 41 to 307 kiloDaltons (Fishman M. L., Chau H. K., Kolpak F., Brady J. 2001. Solvent effects on the molecular properties of pectins. J Agric Food Chem. 49(9):4494-501), native pectins averaging 150 to 200 kD (Catoire, L.; Goldberg, R.; Pierron, M.; Morvan, C.; Herve du Penhoat, C. 1998. An efficient procedure for studying pectin structure which combines limited depolymerization and 13C NMR. European Biophysics Journal, 27(2):127-136). Pectin is composed of three main polysaccharides, polygalacturonan, rhamnogalacturonan I and rhamnogalacturonan II. Rhamnogalacturonan I (rhamnosyl and D-galacturonosyl) sections contain hairy branch points with side chains of arabinose and galactose; while rhamnogalacturonan II side chains contain D-xylose, L-fucose, D-glucuronic acid, D-apiose, 3-deoxy-D-manno-2-octulosonic acid, and 3-deoxy-D-lyxo-2-heptulosonic acid attached to polygalacturonic acid (PGA). Gelling properties of pectins depend on esterification, sugars, pH, water, and temperature. Low methoxyl-pectins gel by di-cation bridging forming egg-box junction zones with Ca2+. Acyl groups prevent gelation with Ca2+; Na+ and K+ non-gelling; and, by nature of carboxylate groups removing Ca2+, gelling by divalent cations is ordered Mg2+<<Ca2+ & Sr2+<Ba2+. High methoxyl-pectins form two-dimensional molecules with sugar, acid, and water immobilized by H-bonds and hydrophobic interactions. In fact, the ratio of bound sugar to pectin is 100 to 1 w/w (Fishman, M. L., Cooke, P. H., Coffin, D. R. Nano Structure Of Native Pectin Sugar Acid Gels Visualized By Atomic Force Microscopy. Biomacromolecules. 2004. 5(2), 334-341).
The potential for pectin to be modified within the cell wall is evidenced by observations that its bilayered organization remains intact after extraction by water and 8 M urea (Bacic, A. and Stone, B. A. 1981. Chemistry and Organization of Aleurone Cell Wall Components from Wheat and Barley. Australian Journal of Plant Physiology 8(5):475-495). Pectin embedded proteins are integral to catalytic chemical reactions essential to covalent bonds and conformation of electrostatic binding of specific enzymes are ruled by interaction only in the presence of Ca2+ (Carpin, S.; Crèvecoeur, M.; de Meyer, M.; Simon, P.; Greppin, H.; and Penel, C. 2001. Identification of a Ca2+-Pectate Binding Site on an Apoplastic Peroxidase. Plant Cell, Vol. 13, 511-520); therefore, Ca2+ maintains ionic bonds in pectin which change in the living system. Understanding and novel assembly of the above characteristics of pectin were the bases for development of the present invention of in vivo structuring of pectin with Ca2+-chelator and saccharide compositions for penetration of exogenous compounds through molecular integration.
Pectin is charged with PGA-chains bearing evenly distributed negative charges; pectin is, thereby, suitable for interactions with positively charged molecules. Ca2+-chelators and their cations, especially calcium itself, are the most appropriate candidates for interaction. This feature has proven highly beneficial to methods and compositions of the present invention to transport of any of a variety of nutrients and active ingredients into pectic cells for immediate metabolism.
Specifically, methods and compositions of the present invention include a selection of Ca2+-chelators as vehicles for extraction of pectins that motivate interactions of components into cells with a selection of saccharides. In the course of the present invention, chelactant compositions of exogenous sugars and Ca2+-chelators develop molecular bonds not only as part of pectin, but also as major exchangeable components of pectin that determine what enters the cell and how weak or strong the bonds will be that allow active transport to occur. According to the concept of the present invention, by designing the normal 100:1 ratio of sugar to pectin to a greater proportion through an import of sugars, the pectin molecule may be enlarged. Add to that, cations in water, and an enhanced number of hydrogen bonds, ionic bonds, and hydrophobicity develop with the integration of exogenous sugars, water and cations into the living cell. For the most part, cations form ionic bonds with pectin. Ionic bonds being some of the weakest of molecular bonds, leave the exogenous cations fully capable of moving internally and throughout an organism by, for example, active transport. Ions are imported and exchanged; urea and ammoniacal nitrogen can be pulled into the cell; and prizing water in pectin gels enhances water-holding capacity of live cells resulting in vigorous enhancement of turgidity for the whole plant. Treatment of live cells with appropriate concentrations of Ca2+-chelators in compositions of the present invention causes an ephemeral nanoscalar draw on calcium within pectin, forcing the entire pectin molecule to change. When exogenous chemicals are made available at that time of extraction, ionic, hydrogen and covalent bonds may develop with those compounds. Bonds, however weak or strong, make the exogenous compounds part of the newly formed pectin molecule.
U.S. Pat. No. 5,767,378 provides a method for selecting genetically engineered eukaryotic cells for mannose or xylose metabolism and demonstrated experimentally that xylose is not metabolized as a carbohydrate source. This gives certain indication that, a saccharide composition, of itself, is not an effective penetrant primarily because there is no natural route of metabolism. Recent genetic sequencing reports disclose proteins that code for synthesis of plant recognition or signaling components. However, insertion of genes for synthesis of human sugars in plants (Fujiyama et al. 2005. Plant cell having animal-type sugar chain adding function. U.S. patent application Ser. No. 20050144670 Kind Code A1); and identification of proteins that regulate proton pumps (Li, J.; Yang, H.; Peer, W. A.; Richter, G.; Blakeslee, J.; Bandyopadhyay, A; Titapiwantakun, B; Undurraga, S.; Khodakovskaya, M.; Richards, E. L.; Krizek, B.; Murphy, A. S.; Gilroy, S.; and Gaxiola, R. 2005. Arabidopsis H+-PPase AVP1 Regulates Auxin-Mediated Organ Development. Science 7 October 2005:121-125), in this class of genetic manipulation bears no relation to in vivo manipulation of pectins of the present invention. Properties of commercial pectin have been characterized based on utilization in food and drug processing and procedures for observing depolymerization (Catoire, L.; Goldberg, R.; Pierron, M.; Morvan, C.; Hervédu Penhoat, C. 1998. An efficient procedure for studying pectin structure which combines limited depolymerization and 13C NMR. European Biophysics Journal, Volume 27, Number 2, Pages: 127-136) to loading pectin clusters with pharmaceuticals (Kun Cheng and Lee-Yong Lim. 2004. Insulin-Loaded Calcium Pectinate Nanoparticles: Effects of Pectin Molecular Weight and Formulation pH. Drug Development and Industrial Pharmacy, Volume 30, Number 4:359-367) have been developed; and, as well, manipulation of the gel in food processing, see for example, United States Patent Application 20010003596 Kind Code A1 (Finnie, K. J.; Olsen, R. J.; Frinak, S. C. Jun. 14, 2001. Multi-stage thickening composition for use with packaged food items and process for using same), further evidencing a need for modified pectins; however, prior in vivo manipulation of pectin by application of sugars is not evident. The compositions of the present invention offer systems under which pectins may now be designed and created in a manner that will be of benefit to food and pharmaceutical industries.
Sugars have not been utilized directly as surfactants, but derivative polymers have, as, for example, U.S. Pat. No. 6,440,907 polyoxyethylene derivative of methyl glucoside; and U.S. Pat. No. 6,746,988 alkylpolyglycoside-derivative benoxacor, cloquintocet, dichlormid, fenclorim, fluxofenime, furilazole, and oxabetrinil; and U.S. Pat. No. 6,826,866 alkylpolyglycosidic surfactant-fertilizer.
Methyl glucoside is a natural metabolite in roses that drives buds to senescence (Ichimura, K.; Mukasa, Y.; Fujiwara, T.; Kohata, K.; Goto, R.; and Suto, K. 1999. Possible roles of methyl glucoside and myo-inositol in the opening of cut rose flowers. Annals of Botany 83:551-557). U.S. Pat. Nos. 5,549,729, 5,797,976, 6,309,440 and 6,318,023 claim utilization of molasses for plant growth promotion. U.S. Pat. No. 5,958,104 utilizes alkyl glucoside as a plant growth regulator; and U.S. Pat. No. 6,358,293 applies methyl glucoside with 20 ppm to 75 ppm manganese, far beyond the range of manganese of the present invention. Polyacylglycosides and polyalkylglycosides of U.S. Pat. No. 6,258,749 enhance plant growth, but do not claim Ca2+; -chelators. U.S. Statutory Invention Registrations Nos. H224 and H303 described R(OG)x plant growth regulatory alkylpolyglycosides. U.S. Pat. No. 6,544,511 describes inoculating Streptomyces sp. R-5 to a plant for disease resistance wherein xylose is a minor bacterial substrate. Prior art for a Ca2+-chelator as saccharide transfer agent and integrator does not exist. For the most part, reference to complexing agents of the prior art is for the acid; whereas, the Ca2+-chelators of the present invention are selected from ammonium, diammonium, potassium, dipotassium, tripotassium, sodium and disodium, salts, and the like.
Without limitation to the case at hand, an example considering transfer of major nutrients and micronutrients into plant systems as a consequence of penetration by means of Ca2+-chelators is provided. Nutrient deficiency leads to a loss of plant productivity. Plants deficient in a particular element will exhibit symptoms that usually reflect specific elemental limitation. Common symptoms of nutrient deficiency are chlorosis and necrosis. A deficiency of potassium results in growth retardation. Deficiency of zinc causes little leaf. Iron deficiency is indicated by chlorosis in new shoots. Boron deficiency results in bronzing and loss of meristematic growth. Phosphorus deficiency makes leaves turn purple. When such symptoms are exhibited in the field, the crop is curable by supplementation with minerals. In alkaline environments, many minerals may be present, but precipitation makes the metals drop out of solution rendering them unavailable. Incomplete penetration leaves residues at the surface that, upon evaporation of the carrier, may reach toxic concentrations leading to damage. The key is to make the mineral supplement available for metabolism and, although maintaining solubility of metals is most often accomplished through sequestration, conventional agents are not built to penetrate being non-functional within the alkaline boundaries of soaps that are meant to penetrate. U.S. Pat. Nos. 5,688,981, 5,962,717 and 5,993,504 originate chelactants, ethylenediaminetriacetic acid (ED3A), that are the mildest of their class. A novel class of chemical compound of the present invention, the macrochelactant (Mac), solves the problems of the prior art. When dissolved in water and applied to the phylloplane, this embodiment of the present invention comprises Macs with concentrations of sequestered metals that penetrate efficiently for recovery of a plant from nutrient deficiency. Biologically required metals include potassium, calcium, magnesium, iron, boron, cobalt, copper, manganese, molybdenum, silicon, zinc and nickel; and of these, magnesium and iron are of particular importance, namely, because soluble forms are often depleted. Magnesium precipitates out of alkaline waters and soils. When soluble, magnesium may be incorporated into a chlorophyll molecule. Soluble iron may be utilized for energy transport chains and, in plants, pigmentation improves. One of the major benefits of nutrient supplementation through a Mac is penetration of the entire dose in a manner that leaves no harmful residue on the surface. The complete penetration of, for example, a di-cationic magnesium-Mac results in robust photosynthetic energy transfer and prosperous yields. With particular reference to iron, it is known by convention of prior art that high concentrations, above 1 ppm, are often lethal to foliage, especially when residual iron remains on the surface. Iron and magnesium to facilitate chlorophyll synthesis has, until now, been limited to exceedingly limited exposure, but can be accomplished by Mac processes and compositions of the present invention at many times the conventional dose.
Agricultural formulations contain additives, but prior additives have not been designed from nanotechnological architecture. The utilization of pectins to integrate desired compounds into the cells of an organelle is novel and has broad application throughout the field because molecular incorporation provides an efficient mechanism for transporting desired products into a living system. Exogenous saccharides, of themselves, cannot penetrate foliar cells. Instead, sugars dry into a shiny, sticky surface film on leaves and on exoskeletons of pests. The sugars of the side chains of pectins, i.e., apiose, arabinose, 3-deoxy-D-manno-2-octulosonic acid, 3-deoxy-D-lyxo-2-heptulosonic acid, fucose, galactose, glucuronic acid, and xylose are abundant in cell walls of green plants; and therefore, processes of the present invention mine vegetative waste materials for raw materials. When utilized in surface coatings that do not integrate with cells, specific functions may be blocked by the aforementioned sugars of vegetative waste, providing for exemplary embodiments of the present invention as, for example, growth retardant and anti-transpirant.
U.S. Pat. No. 6,407,040 applied chitin with mannitol to reduce transpiration, but because growth was inhibited by the intact anti-transpirant, U.S. Pat. No. 6,464,995 later enclosed kaolin particulates in the same membrane to allow gas exchange. The use of anti-transpirants has been researched, e.g., Rao, N. K. S. 1985. The Effects of Antitranspirants on Leaf Water Status, Stomatal Resistance and Yield in Tomato, J Hort Sci 60:89-92; Rajan, M. S.; Reddy, K. R.; Rao, R. S.; Reddi, G. H. S. 1981. Effect of Anti-transpirants and Reflectants on Pod Yield of Rainfield Groundnut. Agri Sci Dig 1:205-206; Kamp, M. 1985. Control of Erysiphe cichoracearum on Zinnia elegans, with a Polymer-based Anti-transpirant. Hort. Sci. 20:879-881; Zekaria-Oren, J. and Eyal, Z. 1991. Effect of Film forming Compounds on the Development of Leaf Rust on Wheat Seedlings. Plant Dis. 75:231-234), and the utilization of any prior anti-transpirant is generally regarded as undesirable because, inherent to its function of blocking evapotranspiration, anti-transpirant films reduce exchange of photosynthetic gases through living plants. Decreased yields have long been directly correlated to the reduction of assimilation inhibiting growth. Clearly, a means of reestablishing normal evapotranspiration would be beneficial to crop management.
A discovery of the present invention solves the problem of lack of penetration of saccharides. On application of Ca2+-chelators to saccharides on a pectin surface, the following events of the present invention are observed:
1) Ca2+-chelators draw out structural Ca2+ and pectin mildly and to a limited extent; 2) Exogenous sugars are pulled in; and 3) Exogenous sugars (and any exogenous cations) bind to pectin. Exogenous saccharides, thereby, not only become part of pectin, they penetrate into the organelle as they do so. As the Ca2+-chelator-saccharide is incorporated into the cell, molecular integration is thereby, broadly applicable to any appropriate composition of matter that may be carried along with it into a pectic cell including, without limitation or exclusion of other compounds, active ingredients, inert ingredients, nutrients, and additives. Based on this integrative approach, a novel aspect of the present invention is that after application of a coating that can function as, for example an anti-transpirant, its inhibitory functions are, for the first time, reversible by [applying] molecular integration compositions of the present invention. With foresight, surface coatings may now be designed to penetrate by serial applications of compositions of the present invention.
A reversal mechanism, hereinafter, protranspirant, composition of the present invention that is biodegradable into a cell would be most beneficial and is, therefore, introduced as another embodiment of the present invention. These embodiments of the present invention relate to methods for blocking growth; coatings; anti-transpirant compositions; penetrant saccharides; and protranspirant compositions.
Wilt is symptomatic of a need for a remedy that prevents total crop loss to death, but treatment with an anti-transpirant has been regarded as a drastic measure to take because when transpiration stops, productivity comes to a standstill. Anti-transpirants often coat foliage without penetration to reduce transpirational water loss mechanically. A means of resuming functions to minimize losses would be most beneficial to growers. In a method of the present invention, compositions are applied to shoots of a plant in an effective amount and with a frequency sufficient to retard growth and prevent wilting and then followed by methods that return evapotranspiration to normal function. Compositions and methods of the present invention return the health and yield of crops to their original state by freeing the plant from the drastic effects of conventional anti-transpirants. Exploitation of the growth retardant properties of coatings are also put to benefit by adjusting formulas to retain slowed growth to reduce maintenance without weakening, such as in golf greens; and as a means of preparing a plant towards optimized pesticide response, such as by weakening a weed prior to application of a herbicide. It would be beneficial to buyers to provide these safe compositions from natural products. As pectic components are vegetative, see for example, Decreux and Messiaen 2005 (Wall-associated Kinase WAK1 Interacts with Cell Wall Pectins in a Calcium-induced Conformation Plant and Cell Physiology 46(2):268-278), processes of resourcing waste are also offered.
It would be beneficial to growers to provide safe compositions that integrate nutrients and active ingredients into live cells, making compositions more highly available for plants than previously afforded; that reduce toxicity of essential minerals, additives and active ingredients while making them available; and that penetrate, transfer, transport, spread, emulsify, wet, stick, safen, metabolize, blend and provide other utilizations of active ingredients by entry for greater purpose. The present invention seeks to fulfill these needs and provides further related advantages.